Regulation of methane production by methanogenic bacteria has several important agronomic and environmental utilities. It has long been recognized that the regulation of methane production in cattle rumen has affected the efficiency with which cattle produce milk and beef from feedstocks. Additionally, there has been renewed environmental interest in the regulation of methane as a major greenhouse gas.
Microbial methane formation is a strictly anaerobic process which is carried out by a metabolically unique group of organisms generally known as the methanogenic bacteria. The group comprises the genera Methanococcus, Methanobacterium, Methanosarcina, Methanobrevibacter, Methanothermus, Methanothrix, Methanospirillum, Methanomicrobium, Methanococcoides, Methanogenium, and Methanoplanus. These bacteria are widely distributed in strictly anaerobic habitats including the rumen of ruminant animals, the termite gut, landfills, stagnant ponds, anaerobic digestors and rice paddies. The temperature range for growth may range from mesophilic temperatures up to extremely thermophilic temperatures.
The methanogens are highly interactive ecologically, and depend heavily on the metabolism of other bacteria to produce the substrates needed for their survival. Fermentative bacteria provide these substrates by conversion of complex macromolecules such as cellulose or protein into four principal methanogenic substrates: hydrogen, carbon dioxide, acetic acid, and formic acid. The methanogens then remove these fermentative end-products and convert them into gaseous methane and carbon dioxide.
The classic example of this type of association is termed "interspecies hydrogen transfer" wherein a hydrogen-producing organism generates hydrogen for the methanogen, and the methanogen then removes hydrogen which is actually inhibitory for the hydrogen producer. This is seen in the natural food chain where primary bacteria convert cellulose to various products including lactate, acetate, fatty acids, carbon dioxide and hydrogen, and the methanogens then utilize the hydrogen and carbon dioxide to produce methane and water.
In marine or brackish waters where sulfate is abundant, cellulose is converted to carbon dioxide and hydrogen sulfide by sulfate reducing bacteria (SRB). These bacteria have a parallel metabolism to the methanogens and are able to utilize hydrogen and sulfate to produce hydrogen sulfide. In sewage treatment facilities and in freshwater bogs where sulfate concentrations are low, the SRB enter into a symbiotic relationship with the methanogens wherein the SRB produce hydrogen from organic acids and alcohols. The methanogens in turn convert the hydrogen to methane and carbon dioxide.
Even though methanogens are typically grown in the laboratory under an 80%/20% (vol/vol) hydrogen/carbon dioxide, in natural environments methanogens and SRB are exposed to and grow on only traces of hydrogen and carbon dioxide. The intermediary levels of hydrogen, carbon dioxide and acetate may be very low but the methanogens and sulfate-reducers are able to grow on these substrates liberated by the fermentation of sugars, organic acids (i.e., lactate, fatty acids) and alcohols.
There are at least two important utilities for inhibitors of methanogenesis. The first is the chemical manipulation of rumen fermentation as it occurs in ruminant animals such as cows and sheep, to divert microbial rumen metabolism away from methane formation and toward volatile fatty acid formation. Methane represents a caloric loss to the ruminant of 5-10% of its total caloric intake, and diversion of this energy into volatile fatty acids which the ruminant would use for nutrition would increase the efficiency of conversion of feedstocks into beef. An inverse relationship between methane formation and production of the volatile fatty acid, propionate, has been demonstrated by many investigators, and therefore a positive effect of a methane inhibitor on rumen nutrition is expected. (C. J. Van Nevel, D. I. Demeyer, Manipulation of rumen fermentation. In: The Rumen Microbial Ecosystem, P. N. Hobson. (ed) Elsevier Publishing Co. (1988).)
Another important application of the inhibition of methane formation would be a decrease in production of a major greenhouse gas and atmospheric pollutant.
Although methane constitutes only 0.4% of all greenhouse pollutants, it contributes 18% of the total greenhouse warming of the earth's atmosphere, and its annual rate of increase is on the order of 1%. Some of the primary sources of environmental methane come from domestic animals, landfills, and rice cultivation; which together contribute over 40% of the total methane emissions and over 60% of the anthropogenic methane emissions. Methane emissions from rice cultivation are estimated to contribute about 20% of the total methane produced in the atmosphere, and emissions form landfills constitute about 7% of the total emissions. With respect to animal methane production, cattle are the ruminants primarily responsible for the largest methane emissions. The average dairy cow may produce 200 liters of methane per day. The U.S. herd alone produces over 5 million metric tons of methane per year. Thus, the agricultural and industrial activities of man have become a significant contributor to the total methane emission into the earth's atmosphere.
Methanogen inhibitors have been developed previously, primarily for use as feedstock additives to increase ruminant efficiency. Such additives fall primarily into two classes. The first group are compounds which indirectly affect methane formation by interfering with carbon or electron flow at a point upstream of the methanogen in the microbial food chain. The second group affects methanogens directly. Examples of compounds known to inhibit methanogenesis directly or indirectly are diverse, and range from common anions such as nitrate, to ionopore antibiotics. Specific examples include monesin, lasalocid, salinomycin, avoparcin, aridcin, actaplanin, penicillin, chlorine and bromine methane analogs, long chain fatty acids, sulfate and nitrate. A complete list is cited in C. J. Van Nevel, D. I. Demeyer, Manipulation of Rumen Fermentation, In: The Rumen Microbial Ecosystem, P. N. Hobson (ed) Elsevier Publishing Co. (1988) hereby incorporated by reference. Clearly most, if not all, of these compounds lack specificity for methane formation, and some exhibit a multitude of side effects in the rumen of animals.
Numerous patents have been granted on a variety of compounds claiming to directly or indirectly inhibit methane formation in ruminant animals. It is believed that none, however, disclose use of anthracquinones as inhibitors of methane production.
The biological activities of anthraquinones are multitudinous and the utility of these compounds includes, for example, use as antimicrobials, proteolytic enzyme inhibitors, and as laxatives. The antimicrobial activity of anthraquinone plant extracts such as Cassia sp. has been long recognized. The active component of Cassia has been identified as 4,5 dihydroxyanthraquinone-2-carboxylic acid (Anchel, J. Biol. Chem., 177:169-177 (1949)). The existing literature, however, indicates that the general antimicrobial effects of anthraquinones appear to be sporadic and unpredictable with regard to the bacterial species and processes affected. Studies have shown, for example, that some gram positive bacterial species such as Bacillus or Staphylococcus are sensitive to anthraquinone, but that gram negative bacteria such as Escherichia sp. or Pseudomonas sp. are insensitive (Kavanaugh, J. Bacteriol., 54:761-767 (1947)). However, other studies have shown that the 1,4,6,8 tetrahydroxyanthraquinone does not inhibit all strains of Bacillus, and that in Nocardia (gram positive) only one strain out of four is effected. The compound has no demonstrated effect on Escherichia coli, Pseudomonas sp., Salmonella sp. or Sarcina sp. (Anke et al., Arch. Microbiol., 126:223-230 (1980); Anke et al., Arch. Microbiol., 126:231-236 (1980)). Metal Chelates of the 1,8 dihydroxyanthraquinone were shown to be active against Bacillus subtilis, Bacillus stearothermophilus and Staphylococcus aureus whereas the 1,2 dihydroxyanthraquinone and the 1-amino-4-hydroxyanthraquinone were generally inactive against these strains. The anthraquinones aloe-emodin and Rhein were found to be inhibitory to Bacillus subtilis and Staphylococcus aureus. However, the related anthraquinone, Chrysophanol, was not inhibitory to these strains. None of the anthraquinones tested inhibited the yeast Candida (Fuzellier et al., Ann. Pharm. Fr., 39(4) 313-318 (1981)). Diaminoanthraquinones were shown to exhibit toxicity against gram positive cocci but not gram negative bacteria (Haran et al., Isr. J. Med. Sci., 17(6): 485-496 (1981)). These results typify the sporadic and unpredicatable antimicrobial effects of the anthraquinones.
Swiss Patent No. 614,466 discloses that anthraquinones with substituent methyl, hydroxymethyl, carboxyl, aldehyde or carboxyethyl groups are known to inhibit bacterial growth in tissue culture and in other applications where eukaryotic growth is desirable, but bacterial growth is not.
The 1,3,6,8 tetrahydroxyanthraquinone has been claimed as producing a laxative effect by stimulation of the neuromuscular junction of the bowel wall (U.S. Pat. No. 5,039,707).
Anthraquinones have also been shown to interfere with bacterial DNA metabolism (Anke et al., Arch. Microbiol., 126:231-236 (1980)); and to inhibit ADP transport into mitochondria (Boos et al., FEBS Lett., 127:40-44 (1981)). The chemical reaction of reduced anthraquinone with oxygen to produce toxic superoxide radical may also be an important toxicity mechanism (Shcherbanoviskii et al., Rastit. Resur., 11(3): 445-454 (1975)).
Miscellaneous inhibitory effects on particular enzyme systems have been reported, but the overall lack of toxicity of anthraquinones is supported by their natural occurrence in plants, their widespread use as vat dyes for clothing and their use until recently as laxatives. Pharmaceutical use of anthraquinones, particularly hydroxylated anthraquinones, has been curtailed due to the finding that they are weak mutagens. Halogenated anthraquinones, however, are not mutagenic (Brown et al., Mutation Research, 40:203-224 (1976)).
U.S. patent application Ser. No. 07/510,763, Pct publication No. 91/15954 discloses that a large number of anthraquinone derivatives inhibit respiratory sulfate-reduction from anaerobic sulfate-reducing bacteria. Further, it was shown that other growth modes within these bacteria were unaffected and that other bacterial types such as Escherichia coli and Saccharomyces sp. were unaffected by the preferred compounds. The preferred anthraquinones comprised halogenated as well as hydroxylated derivatives. These compounds were shown to inhibit sulfide production in all laboratory strains of sulfate-reducing bacteria, as well as crude sulfate-reducing enrichments from a variety of natural environments.
In summary, although it has been shown that anthraquinones possess a variety of rather specific biological properties, these compounds have never before been implicated as inhibitors of the methanogenic process. Use of these compounds fills a need, therefore, as inhibitors of methane production from methanogenic bacteria. Preferably, this inhibition should be generally non-toxic, and have the ability to inhibit methane production without significantly disrupting the natural equilibrium of the existing microbial population.